Scleroderma (SSc) is a serious disease of unknown cause characterized by excessive accumulation of collagen and other connective tissue components in skin and internal organs. The mechanisms responsible for such accumulation are not known. In previous work from our laboratories we demonstrated that the Tsk/+ (tight skin) mutant mouse displays connective tissue abnormalities that closely resemble those present in patients with SSc, including increased collagen tissue deposition, and increased biosynthesis in skin organ cultures and cultured fibroblasts. Furthermore, we demonstrated a coordinated increase in the levels of type I and type III collagen mRNAs in these cells. These results suggest that the Tsk mice are an ideal experimental model for the study of the connective tissue alterations in SSc. The purpose of the work proposed in this application will be to continue our in-depth studies of the mechanisms responsible for the connective tissue alterations displayed by Tsk mice at the molecular level applying state of the art recombinant DNA techniques currently used in our laboratories. We will identify the regulatory defect in Tsk mice by mapping pertinent regions of type I and type VI collagen genes by transient transfections of intact and deleted putative regulatory sequences of their corresponding promoters into normal and Tsk/+ fibroblasts and into embryonic cell lines that are homozygous for the Tsk mutation. Because of the likely importance of the Tsk gene in the regulation of collagen gene expression under normal conditions and in fibrotic diseases such as SSc, we have initiated studies to identify the location and to clone the Tsk gene employing intersubspecific backcross studies and chromosome walking. The function of product(s) of potential candidate genes from the Tsk region will be identified and the identity of the Tsk gene will be proved employing transgenic mice. Biosynthesis and regulation of collagen in dermal fibroblasts from the newly described Tsk-2 mutant will be initiated to determine similarities and differences with those in Tsk/+ fibroblasts and embryonic Tsk/Tsk cell lines and to determine the molecular basis of the defect. It is expected that the knowledge gained from these studies will be of direct relevance to understand the pathogenesis of the excessive collagen deposition characteristic of SSc and will provide a more rational approach to develop possible modes of therapy for this incurable and devastating disease.